Method for treating diseases using HSP90-inhibiting agents in combination with antimitotics

ABSTRACT

The present invention provides a method for treating cancer. The method involves the administration of an HSP90 inhibitor and an antimitotic, where the combined administration provides a synergistic effect. In one aspect of the invention, a method of treating cancer is provided where a subject is treated with a dose of an HSP90 inhibitor in one step and a dose of an antimitotic in another step. In another aspect of the invention, a method of treating cancer is provided where a subject is first treated with a dose of an HSP90 inhibitor and subsequently treated with a dose of an antimitotic. In another aspect of the invention, a method of treating cancer is provided where a subject is first treated with a dose of an antimitotic and subsequently treated with a dose of an HSP90 inhibitor.

CROSS REFERENCE TO RELATED U.S. PATENT APPLICATIONS

The present application claims the benefit of Provisional PatentApplication No. 60/474,906, which was filed May 30, 2003, under 35U.S.C. § 119(e). The provisional application is herebyincorporated-by-reference into this application for all purposes.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to methods for treating cancer in which aninhibitor of Heat Shock Protein 90 (“HSP90”) is combined with anantimitotic. More particularly, this invention relates to combinationsof the HSP90 inhibitor geldanamycin and its derivatives, especially17-alkylamino-17-desmethoxygeldanamycin (“17-AAG”) and17-(2-dimethylaminoethyl)amino-17-desmethoxygeldanamycin (“17-DMAG”),with an antimitotic (e.g., docetaxel, discodermolide, vinblastine,vincristine, vindesine, and epothilone D).

REFERENCES

-   Agnew et al., “Clinical pharmacokinetics of    17-(allylamino)-17-demethoxygeldanamycin and the active metabolite    17-(amino)-17-demethoxygeldanamycin given as a one-hour infusion    daily for 5 days.” AACR, 2002.-   An et al., “Depletion of p185erbB2, Raf-1 and mutant p53 proteins by    geldanamycin derivatives correlates with antiproliferative    activity.” Cancer Chemother. Pharmacol. 40:60-64, 1997.-   Bagatell et al., “Induction of a heat shock factor 1-dependent    stress response alters the cytotoxic activity of hsp90-binding    agents.” Clin. Cancer Res. 6:3312-3318, 2000.-   Bagatell et al., “Destabilization of steroid receptors by heat shock    protein 90-binding drugs: a ligand-independent approach to hormonal    therapy of breast cancer.” Clin. Cancer Res. 7:2076-2084, 2001.-   Banerji et al., “A pharmacokinetically (PK)-pharmacodynamically (PD)    driven Phase I trial of the HSP90 molecular chaperone inhibitor    17-allylamino-17-demethoxygeldanamycin (17-AAG).” AACR, 2002.-   Barent et al., “Analysis of FKBP51/FKBP52 chimeras and mutants for    Hsp90 binding and association with progesterone receptor complexes.”    Mol. Endocrinol. 12:342-354, 1998.-   Bilodeau et al., “Tyrosine kinase inhibitors.” U.S. Pat. No.    6,245,759 issued Jun. 12, 2001.-   Citri et al., “Drug-induced ubiquitylation and degradation of ErbB    receptor tyrosine dinases: implications for cancer chemotherapy.”    EMBO Journal 21:2407-2417, 2002.-   Egorin et al., “Metabolism of    17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murine and    human hepatic preparations.” Cancer Res. 58:2385-2396, 1998.-   Fraley et al., “Tyrosine kinase inhibitors.” U.S. Pat. No. 6,306,874    issued Oct. 23, 2001.-   Fraley et al., “Tyrosine kinase inhibitors.” U.S. Pat. No. 6,313,138    issued Nov. 6, 2001.-   Gaidigk et al., “NAD (P)H:quinone oxidoreductase: polymorphisms and    allele frequencies n Caucasian, Chinese and Canadian Native Indian    and Inuit populations.” Pharmacogenetics 8:305-313, 1998.-   Gelmon et al., “Anticancer agents targeting signaling molecules and    cancer cell environment: challenges for drug development?” J. Natl.    Cancer Inst. 91:1281-1287, 1999.-   Goetz et al., “The Hsp90 chaperone complex as a novel target for    cancer therapy.” Ann. Oncol. 14:1169-1176, 2003.-   Goh et al., “Explaining interindividual variability of docetaxel    pharmacokinetics and pharmacodynamics in Asians through phenotyping    and genotyping strategies.” J. Clin. Oncol. 20:3683-3690, 2002.-   Grenert et al., “The amino-terminal domain of heat shock protein 90    (hsp90) that binds geldanamycin is an ATP/ADP switch domain that    regulates hsp90 conformation.” J. Biol. Chem. 272:23843-23850, 1997.-   Johnson and Toft, “Binding of p23 and hsp90 during assembly with the    progesterone receptor.” Mol. Endocrinol. 9:670-678, 1995.-   Hartl and Hayer-Hartl, “Molecular chaperones in the cytosol: from    nascent chain to folded protein.” Science 195:1852-1858, 2002.-   Hegde et al., “Short circuiting stress protein expression via a    tyrosine kinase inhibitor, herbimycin A.” J. Cell Physiol.    165:186-200, 1995.-   Hustert et al., “The genetic determinants of the CYP3A5    polymorphism.” Pharmacogenetics 11:773-779, 2001.-   Kelland et al., “DT-Diaphorase expression and tumor cell sensitivity    to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat    shock protein 90.” J. Natl. Cancer Inst. 91:1940-1949, 1999.-   Kuehl et al., “Sequence diversity in CYP3A promoters and    characterization of the genetic basis of polymorphic CYP3A5    expression.” Nat. Genet. 27:383-391, 2001.-   Lawson et al., “Geldanamycin, an hsp90/GRP94-binding drug, induces    increased transcription of endoplasmic reticulum (ER) chaperones via    the ER stress pathway.” J. Cell Physiol. 174:170-178, 1998.-   Lin et al., “Co-regulation of CYP3A4 and CYP3A5 and contribution to    hepatic and intestinal midazolam metabolism.” Mol. Pharmacol.    62:162-172, 2002.-   Morimoto et al., “The heat-shock response: regulation and function    of heat-shock proteins and molecular chaperones.” Essays Biochem    32:17-29, 1997.-   Munster et al., “Phase I trial of    17-(allylamino)-17-demethoxygeldanamycin (17-AAG) in patients with    advanced solid malignancies.” Proc. Am. Soc. Clin. Oncol, 83a, 2001.-   Munster et al., “Modulation of Hsp90 function by ansamycins    sensitizes breast cancer cells to chemotherapy-induced apoptosis in    an RB- and schedule-dependent manner.” Clin. Cancer Res.    7:2228-2236, 2001.-   Murakami et al., “Induction of hsp 72/73 by herbimycin A, an    inhibitor of transformation by tyrosine kinase oncogenes.” Exp. Cell    Res. 195:338-344, 1991.-   Pratt and Toft, “Steroid receptor interactions with heat shock    protein and immunophilin chaperones.” Endocr. Rev. 18:306-60, 1997.-   Prodromou et al., “Identification and structural characterization of    the ATP/ADP-binding site in the Hsp90 molecular chaperone.” Cell    90:65-75, 1997.-   Richter and Buchner, “Hsp90: chaperoning signal transduction.” J.    Cell. Physiol. 188:281-290, 2001.-   Rosvold et al., “Identification of an NAD(P)H:quinone oxidoreductase    polymorphism and its association with lung cancer and smoking.”    Pharmacogenetics 5:199-206, 1995.-   Schneider et al., “Pharmacologic shifting of a balance between    protein refolding and degradation mediated by Hsp90.” Proc. Natl.    Acad. Sci. USA 93:14536-14541, 1996.-   Schnur et al., “erbB-2 oncogene inhibition by geldanamycin    derivatives: synthesis, mechanism of action, and structure-activity    relationships.” J. Med. Chem. 38:3813-20, 1995.-   Schnur et al., “Inhibition of the oncogene product p 185erbB-2 in    vitro and in vivo by geldanamycin and dihydrogeldanamycin    derivatives.” J. Med. Chem. 38:3806-3812, 1995.-   Smith et al., “Progesterone receptor structure and function altered    by geldanamycin, an hsp90-binding agent.” Mol. Cell Biol.    15:6804-6812, 1995.-   Smith et al., “Identification of a 60-kilodalton stress-related    protein, p60, which interacts with hsp90 and hsp70.” Mol. Cell Biol.    13:869-876, 1993.-   Stebbins et al., “Crystal structure of an Hsp90-geldanamycin    complex: targeting of a protein chaperone by an antitumor agent.”    Cell 89:239-250, 1997.-   Traver et al., “NAD(P)H:quinone oxidoreductase gene expression in    human colon carcinoma cells: characterization of a mutation which    modulates DT-diaphorase activity and mitomycin sensitivity.” Cancer    Res. 52:797-802, 1992.-   Whitesell et al., “Inhibition of heat shock protein HSP90-pp60v-src    heteroprotein complex formation by benzoquinone ansamycins:    essential role for stress proteins in oncogenic transformation.”    Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994.-   Young et al., “Hsp90: a specialized but essential protein-folding    tool.” J. Cell Biol. 154:267-273, 2001.-   Zou et al., “Repression of heat shock transcription factor HSF1    activation by HSP90 (HSP90 complex) that forms a stress-sensitive    complex with HSF1.” Cell 94:471-480, 1998.

Discussion

Geldanamycin (figure below, R₁₇=—OCH₃) is a benzoquinone ansamycinpolyketide isolated from Streptomyces geldanus. Although originallydiscovered by screening microbial extracts for antibacterial andantiviral activity, geldanamycin was later found to be cytotoxic tocertain tumor cells in vitro and to reverse the morphology of cellstransformed by the Rous sarcoma virus to a normal state.

Geldanamycin's nanomolar potency and apparent specificity for aberrantprotein kinase dependent tumor cells, as well as the discovery that itsprimary target in mammalian cells is the ubiquitous Hsp90 proteinchaperone, has stimulated interest in the development of this compoundas an anti-cancer drug. However, the association of unacceptablehepatotoxicity with the administration of geldanamycin led to itswithdrawal from Phase I clinical trials.

More recently, attention has focused on 17-amino derivatives ofgeldanamycin, in particular 17-(allylamino)-17-desmethoxygeldanamycin(“17-AAG”, R₁₇=—NCH₂CH═CH₂). This compound has reduced hepatotoxicitywhile maintaining useful Hsp90 binding. Certain other 17-aminoderivatives of geldanamycin, 11-oxogeldanamycin, and5,6-dihydrogeldanamycin, are disclosed in U.S. Pat. Nos. 4,261,989,5,387,584 and 5,932,566, each of which is incorporated herein byreference. Treatment of cancer cells with geldanamycin or 17-AAG causesa retinoblastoma protein-dependent G1 block, mediated by down-regulationof the induction pathways for cyclin D-cyclin dependent cdk4 and cdk6protein kinase activity. Cell cycle arrest is followed bydifferentiation and apoptosis. G1 progression is unaffected bygeldanamycin or 17-AAG in cells with mutated retinoblastoma protein;these cells undergo cell cycle arrest after mitosis, again followed byapoptosis.

The above-described mechanism of geldanamycin and 17-AAG appears to be acommon mode of action among the benzoquinone ansamycins that furtherincludes binding to Hsp90 and subsequent degradation of Hsp90-associatedclient proteins. Among the most sensitive client protein targets of thebenzoquinone ansamycins are the Her kinases (also known as ErbB), Raf,Met tyrosine kinase, and the steroid receptors. Hsp90 is also involvedin the cellular response to stress, including heat, radiation, andtoxins. Certain benzoquinone ansamycins, such as 17-AAG, have thus beenstudied to determine their interaction with cytotoxins that do nottarget Hsp90 client proteins.

U.S. Pat. Nos. 6,245,759, 6,306,874 and 6,313,138, each of which isincorporated herein by reference, disclose compositions comprisingcertain tyrosine kinase inhibitors together with 17-AAG and methods fortreating cancer with such compositions. Münster, et al., “Modulation ofHsp90 function by ansamycins sensitizes breast cancer cells tochemotherapy-induced apoptosis in an RB- and schedule-dependent manner,”Clinical Cancer Research (2001) 7:2228-2236, discloses that 17-AAGsensitizes cells in culture to the cytotoxic effects of Paclitaxel anddoxorubicin. The Münster reference further discloses that thesensitization towards paclitaxel by 17-AAG is schedule-dependent inretinoblastoma protein-producing cells due to the action of these twodrugs at different stages of the cell cycle: treatment of cells with acombination of paclitaxel and 17-AAG is reported to give synergisticapoptosis, while pretreatment of cells with 17-AAG followed by treatmentwith paclitaxel is reported to result in abrogation of apoptosis.Treatment of cells with paclitaxel followed by treatment with 17-AAG 4hours later is reported to show a synergistic effect similar tocoincident treatment.

Citri, et al., “Drug-induced ubiquitylation and degradation of ErbBreceptor tyrosine kinases: implications for cancer chemotherapy,” EMBOJournal (2002) 21:2407-2417, discloses an additive effect uponco-administration of geldanamycin and an irreversible protein kinaseinhibitor, CI-1033, on growth of ErbB2-expressing cancer cells in vitro.In contrast, an antagonistic effect of CI-1033 and anti-ErB2 antibody,Herceptin is disclosed.

Thus, while there has been a great deal of research interest in thebenzoquinone ansamycins, particularly geldanamycin and 17-AAG, thereremains a need for effective therapeutic regimens to treat cancer orother disease conditions characterized by undesired cellularhyperproliferation using such compounds, whether alone or in combinationwith other agents.

SUMMARY OF THE INVENTION

The present invention provides a method for treating cancer. The methodinvolves the administration of an HSP90 inhibitor and an antimitotic,where the combined administration provides a synergistic effect.

In one aspect of the invention, a method of treating cancer is providedwhere a subject is treated with a dose of an HSP90 inhibitor in one stepand a dose of an antimitotic in another step.

In another aspect of the invention, a method of treating cancer isprovided where a subject is first treated with a dose of an HSP90inhibitor and subsequently treated with a dose of an antimitotic.

In another aspect of the invention, a method of treating cancer isprovided where a subject is first treated with a dose of an antimitoticand subsequently treated with a dose of an HSP90 inhibitor.

In another aspect of the invention, a method of treating cancer isprovided where a subject is first treated with a dose of an antimitotic(e.g., docetaxel, vinblastine, vincristine, vindesine, and epothiloneD). After waiting for a period of time sufficient to allow developmentof a substantially efficacious response of the antimitotic, aformulation comprising a synergistic dose of a benzoquinone ansamycintogether with a second sub-toxic dose of the antimitotic isadministered.

In another aspect of the invention, a method of treating cancer isprovided where a subject is treated first with a dose of a benzoquinoneansamycin, and second, a dose of an antimitotic. After waiting for aperiod of time sufficient to allow development of a substantiallyefficacious response of the antimitotic, a formulation comprising asynergistic dose of a benzoquinone ansamycin together with a secondsub-toxic dose of the antimitotic drug is administered.

In another aspect of the invention, a method for treating cancer isprovided where a subject is treated with a dose of an HSP90 inhibitor inone step and a dose of an antimitotic in another step, and where a sideeffect profile for the combined, administered drugs is substantiallybetter than for the antimitotic alone.

In another aspect of the invention, a method for treating breast orcolorectal cancer is provided where a subject is treated with a dose ofan HSP90 inhibitor in one step and a dose of an antimitotic in anotherstep. The HSP90 inhibitor for this aspect is typically 17-AAG, while theantimitotic is usually docetaxel, vinblastine, vincristine, vindesine,or epothilone D. For the treatment of colorectal cancer, the antimitoticis typically administered before the 17-AAG; for the treatment of breastcancer, the antimitotic is typically administered after the 17-AAG.Where the antimitotic is epothilone D, it is oftentimes administeredbefore the 17-AAG for the treatment of breast cancer.

Definitions

“Antimitotic” refers to a drug that inhibits or prevents mitosis orprodrugs thereof, excepting out antibiotics and enzyme inhibitors.Examples of antimitotics include, without limitation, vinblastine,vincristine, vindesine, vinorelbine, paclitaxel, docetaxel,discodermolide, epothilone D, etoposide, and teniposide.

“HSP90 inhibitor” refers to a compound that inhibits the activity ofheat shock protein 90, which is a cellular protein responsible forchaperoning multiple client proteins necessary for cell signaling,proliferation and survival. One class of HSP90 inhibitors is thebenzoquinone ansamycins. Examples of such compounds include, withoutlimitation, geldanamycin and geldanamycin derivatives (e.g.,17-alkylamino-17-desmethoxy-geldanamycin (“17-AAG”) and17-(2-dimethylaminoethyl)amino-17-desmethoxy-geldanamycin (“17-DMAG”).See Sasaki et al., U.S. Pat. No. 4,261,989 (1981) for synthesis of17-AAG and Snader et al., US 2004/0053909 A1 (2004) for synthesis of17-DMAG. In addition to 17-AAG and 17-DMAG, other preferred geldanamycinderivatives are11-O-methyl-17-(2-(1-azetidinyl)ethyl)amino-17-demethoxygeldanamycin(A), 11-O-methyl-17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin(B), and11-O-methyl-17-(2-(1-pyrrolidinyl)ethyl)amino-17-demethoxygeldanamycin(C), whose synthesis is described in the co-pending commonly US patentapplication of Tian et al., Ser. No. 10/825,788, filed Apr. 16, 2004,and in Tian et al., PCT application Ser. No. PCT/U.S. 04/11638, filedApr. 16, 2004; the disclosures of which are incorporated herein byreference. Additional preferred geldanamycin derivatives are describedin Santi et al., US 2003/0114450 A1 (2003), also incorporated byreference.

“MTD” refers to maximum tolerated dose. The MTD for a compound isdetermined using methods and materials known in the medical andpharmacological arts, for example through dose-escalation experiments.One or more patients is first treated with a low dose of the compound,typically about 10% of the dose anticipated to be therapeutic based onresults of in vitro cell culture experiments. The patients are observedfor a period of time to determine the occurrence of toxicity. Toxicityis typically evidenced as the observation of one or more of thefollowing symptoms: vomiting, diarrhea, peripheral neuropathy, ataxia,neutropenia, or elevation of liver enzymes. If no toxicity is observed,the dose is increased about 2-fold, and the patients are again observedfor evidence of toxicity. This cycle is repeated until a dose producingevidence of toxicity is eached. The dose immediately preceding the onsetof unacceptable toxicity is taken as the MTD.

“Side effects” refer to a number of toxicities typically seen upontreatment of a subject with an antineoplastic drug. Such toxicitiesinclude, without limitation, anemia, anorexia, bilirubin effects,dehydration, dermatology effects, diarrhea, dizziness, dyspnea, edema,fatigue, headache, hematemesis, hypokalemia, hypoxia, musculoskeletaleffects, myalgia, nausea, neuro-sensory effects, pain, rash, serumglutamic oxaloacetic transaminase effects, serum glutamic pyruvictransaminase effects, stomatitis, sweating, taste effects,thrombocytopenia, voice change, and vomiting.

“Side effect grading” refers to National Cancer Institute commontoxicity criteria (NCI CTC, Version 2). Grading runs from 1 to 4, with agrade of 4 representing the most serious toxicities.

Combination Therapy

The present invention provides a method for treating cancer. The methodinvolves the administration of an HSP90 inhibitor and an antimitotic,where the combined administration provides a synergistic effect.

Suitable HSP90 inhibitors used in the present invention includebenzoquinone ansamycins. Typically, the benzoquinone ansamycin isgeldanamycin or a geldanamycin derivative. Preferably, the benzoquinoneansamycin is a geldanamycin derivative selected from a group consistingof 17-alkylamino-17-desmethoxy-geldanamycin (“17-AAG”) and17-(2-dimethylaminoethyl)amino-17-desmethoxy-geldanamycin (“17-DMAG”).

Antimitotics employed in the present method include, without limitation,vinblastine, vincristine, vindesine, vinorelbine, paclitaxel, docetaxel,epothilone D, etoposide, and teniposide.

The dose of antimitotic used as a partner in combination therapy with anHSP90 inhibitor (e.g., benzoquinone ansamycin) is determined based onthe maximum tolerated dose observed when the antimitotic is used as thesole therapeutic agent. In one embodiment of the invention, the dose ofantimitotic when used in combination therapy with a benzoquinoneansamycin is the MTD. In other embodiments of the invention, the dose ofantimitotic when used in combination therapy with a benzoquinoneansamycin is between about 1% of the MTD and the MTD, between about 5%of the MTD and the MTD, between about 5% of the MTD and 75% of the MTD,or between about 25% of the MTD and 75% of the MTD.

Use of the benzoquinone ansamycin allows for use of a lower therapeuticdose of an antimitotic, thus significantly widening the therapeuticwindow for treatment. In one embodiment, the therapeutic dose ofantimitotic is lowered by at least about 10%. In other embodiments thetherapeutic dose is lowered from about 10% to 20%, from about 20% to50%, from about 50% to 200%, or from about 100% to 1,000%.

For the treatment of a variety of carcinomas, the recommendedintravenous dose of various antimitotics is as follows: vinblastine (foradults)—administered once/week with an initial dose of 3.7 mg/m², withgraded doses of 5.5, 7.4, 9.25 and 11.1 mg/m² at 7 day intervals;vincristine (for adults)—administered once/week with an initial dose of0.4 to 1.4 mg/m²; vindesine—administered at a dose of 3 mg/m²;vinorelbine—30 mg/m²/week; paclitaxel—135 mg/m² given IV over 3 hoursonce every 3 weeks (metastatic ovarian cancer, advanced ovarian cancer,and AIDS-related Kaposi's sarcoma) and 175 mg/m² given IV over 3 hoursonce every 3 weeks (metastatic breast cancer); docetaxel—60-100 mg/m²given over 1 hour once every 3 weeks; epothilone D—100 mg/m² once perweek; etoposide—50 to 100 mg/m² for 5 days or 100 mg/m² on alternatedays for three doses (testicular cancer) and 50 to 120 mg/m² per dayintravenously for 3 days or 50 mg per day orally for 21 days (small celllung carcinoma); and, teniposide—50 mg/m² per day for 5 days to 165mg/m² per day twice weekly (lymphoblastic leukemia).

The synergistic dose of the benzoquinone ansamycin used in combinationtherapy is determined based on the maximum tolerated dose observed whenthe benzoquinone ansamycin is used as the sole therapeutic agent.Clinical trials have determined an MTD for 17-AAG of about 40 mg/m²utilizing a daily×5 schedule, an MTD of about 220 mg/m² utilizing atwice-weekly regimen, and an MTD of about 308 mg/m² utilizing aonce-weekly regimen. In one embodiment of the invention, the dose of thebenzoquinone ansamycin when used in combination therapy is the MTD. Inother embodiments of the invention, the does of the benzoquinoneansamycin when used in combination therapy is between about 1% of theMTD and the MTD, between about 5% of the MTD and the MTD, between about5% of the MTD and 75% of the MTD, or between about 25% of the MTD and75% of the MTD.

Where the benzoquinone ansamycin is 17-AAG, and the administration ofcompound is weekly, its therapeutic dose is typically between 50 mg/m²and 450 mg/m². Preferably, the dose is between 150 mg/m² and 350 mg/m²,and about 308 mg/m² is especially preferred. Where the administration ofcompound is biweekly (i.e., twice per week), the therapeutic dose of17-AAG is typically between 50 mg/m² and 250 mg/m². Preferably, the doseis between 150 mg/m² and 250 mg/m², and about 220 mg/m² is especiallypreferred.

Where the present method involves the administration of 17-AAG andvinblastine, a dosage regimen involving one or two administrations ofthe combination per week is typical. Tables 1 and 2 below show a numberof vinblastine/17-AAG dosage combinations (i.e., dosage combinations0001 to 0096). TABLE 1 Vinblastine/17-AAG dosage combinations. 30-100100-150 150-200 200-250 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG  0-1 mg/m² 0001 0002 0003 0004 vinblastine  1-2 mg/m² 0005 00060007 0008 vinblastine  2-3 mg/m² 0009 0010 0011 0012 vinblastine  3-4mg/m² 0013 0014 0015 0016 vinblastine  4-5 mg/m² 0017 0018 0019 0020vinblastine  5-6 mg/m² 0021 0022 0023 0024 vinblastine  6-7 mg/m² 00250026 0027 0028 vinblastine  7-8 mg/m² 0029 0030 0031 0032 vinblastine 8-9 mg/m² 0033 0034 0035 0036 vinblastine  9-10 mg/m² 0037 0038 00390040 vinblastine 10-11 mg/m² 0041 0042 0043 0044 vinblastine 11-12 mg/m²0045 0046 0047 0048 vinblastine

TABLE 2 Vinblastine/17-AAG dosage combinations continued. 250-300300-350 350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG  0-1 mg/m² 0049 0050 0051 0052 vinblastine  1-2 mg/m² 0053 00540055 0056 vinblastine  2-3 mg/m² 0057 0058 0059 0060 vinblastine  3-4mg/m² 0061 0062 0063 0064 vinblastine  4-5 mg/m² 0065 0066 0067 0068vinblastine  5-6 mg/m² 0069 0070 0071 0072 vinblastine  6-7 mg/m² 00730074 0075 0076 vinblastine  7-8 mg/m² 0077 0078 0079 0080 vinblastine 8-9 mg/m² 0081 0082 0083 0084 vinblastine  9-10 mg/m² 0085 0086 00870088 vinblastine 10-11 mg/m² 0089 0090 0091 0092 vinblastine 11-12 mg/m²0093 0094 0095 0096 vinblastine

Where the present method involves the administration of 17-AAG andvincristine, a dosage regimen involving one or two administrations ofthe combination per week is typical. Tables 3 and 4 below show a numberof vincristine/17-AAG dosage combinations (i.e., dosage combinations0097 to 0176). TABLE 3 Vincristine/17-AAG dosage combinations. 30-100100-150 150-200 200-250 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG   0-0.2 mg/m² 0097 0098 0099 0100 vincristine 0.2-0.4 mg/m² 01010102 0103 0104 vincristine 0.4-0.6 mg/m² 0105 0106 0107 0108 vincristine0.6-0.8 mg/m² 0109 0110 0111 0112 vincristine 0.8-1.0 mg/m² 0113 01140115 0116 vincristine 1.0-1.2 mg/m² 0117 0118 0119 0120 vincristine1.2-1.4 mg/m² 0121 0122 0123 0124 vincristine 1.4-1.6 mg/m² 0125 01260127 0128 vincristine 1.6-1.8 mg/m² 0129 0130 0131 0132 vincristine1.8-2.0 mg/m² 0133 0134 0135 0136 vincristine

TABLE 4 Vincristine/17-AAG dosage combinations continued. 250-300300-350 350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG   0-0.2 mg/m² 0137 0138 0139 0140 vincristine 0.2-0.4 mg/m² 01410142 0143 0144 vincristine 0.4-0.6 mg/m² 0145 0146 0147 0148 vincristine0.6-0.8 mg/m² 0149 0150 0151 0152 vincristine 0.8-1.0 mg/m² 0153 01540155 0156 vincristine 1.0-1.2 mg/m² 0157 0158 0159 0160 vincristine1.2-1.4 mg/m² 0161 0162 0163 0164 vincristine 1.4-1.6 mg/m² 0165 01660167 0168 vincristine 1.6-1.8 mg/m² 0169 0170 0171 0172 vincristine1.8-2.0 mg/m² 0173 0174 0175 0176 vincristine

Where the present method involves the administration of 17-AAG andvindesine, a dosage regimen involving one or two administrations of thecombination per week is typical. Tables 5 and 6 below show a number ofvindesine/17-AAG dosage combinations (i.e., dosage combinations 0177 to0256). TABLE 5 Vindesine/17-AAG dosage combinations. 30-100 100-150150-200 200-250 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG 17-AAG  0-0.3 mg/m² 0177 0178 0179 0180 vindesine 0.3-0.6 mg/m² 0181 0182 01830184 vindesine 0.6-0.9 mg/m² 0185 0186 0187 0188 vindesine 0.9-1.2 mg/m²0189 0190 0191 0192 vindesine 1.2-1.5 mg/m² 0193 0194 0195 0196vindesine 1.5-1.8 mg/m² 0197 0198 0199 0200 vindesine 1.8-2.1 mg/m² 02010202 0203 0204 vindesine 2.1-2.4 mg/m² 0205 0206 0207 0208 vindesine2.4-2.7 mg/m² 0209 0210 0211 0212 vindesine 2.7-3.0 mg/m² 0213 0214 02150216 vindesine

TABLE 6 Vindesine/17-AAG dosage combinations continued. 250-300 300-350350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG 17-AAG  0-0.3 mg/m² 0217 0218 0219 0220 vindesine 0.3-0.6 mg/m² 0221 0222 02230224 vindesine 0.6-0.9 mg/m² 0225 0226 0227 0228 vindesine 0.9-1.2 mg/m²0229 0230 0231 0232 vindesine 1.2-1.5 mg/m² 0233 0234 0235 0236vindesine 1.5-1.8 mg/m² 0237 0238 0239 0240 vindesine 1.8-2.1 mg/m² 02410242 0243 0244 vindesine 2.1-2.4 mg/m² 0245 0246 0247 0248 vindesine2.4-2.7 mg/m² 0249 0250 0251 0252 vindesine 2.7-3.0 mg/m² 0253 0254 02550256 vindesine

Where the present method involves the administration of 17-AAG andvinorelbine, a dosage regimen involving one or two administrations ofthe combination per week is typical. Tables 7 and 8 below show a numberof vinorelbine/17-AAG dosage combinations (i.e., dosage combinations0257 to 0336). TABLE 7 Vinorelbine/17-AAG dosage combinations. 30-100100-150 150-200 200-250 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG  0-3 mg/m² 0257 0258 0259 0260 vinorelbine  3-6 mg/m² 0261 02620263 0264 vinorelbine  6-9 mg/m² 0265 0266 0267 0268 vinorelbine  9-12mg/m² 0269 0270 0271 0272 vinorelbine 12-15 mg/m² 0273 0274 0275 0276vinorelbine 15-18 mg/m² 0277 0278 0279 0280 vinorelbine 18-21 mg/m² 02810282 0283 0284 vinorelbine 21-24 mg/m² 0285 0286 0287 0288 vinorelbine24-27 mg/m² 0289 0290 0291 0292 vinorelbine 27-30 mg/m² 0293 0294 02950296 vinorelbine

TABLE 8 Vinorelbine/17-AAG dosage combinations continued. 250-300300-350 350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG  0-3 mg/m² 0297 0298 0299 0300 vinorelbine  3-6 mg/m² 0301 03020303 0304 vinorelbine  6-9 mg/m² 0305 0306 0307 0308 vinorelbine  9-12mg/m² 0309 0310 0311 0312 vinorelbine 12-15 mg/m² 0313 0314 0315 0316vinorelbine 15-18 mg/m² 0317 0318 0319 0320 vinorelbine 18-21 mg/m² 03210322 0323 0324 vinorelbine 21-24 mg/m² 0325 0326 0327 0328 vinorelbine24-27 mg/m² 0329 0330 0331 0332 vinorelbine 27-30 mg/m² 0333 0334 03350336 vinorelbine

Where the present method involves the administration of 17-AAG andpaclitaxel, a dosage regimen involving one or two administrations of thecombination per week or longer (e.g., every 3 weeks) is typical. Tables9 and 10 below show a number of paclitaxel/17-AAG dosage combinations(i.e., dosage combinations 0337 to 0408). TABLE 9 Paclitaxel/17-AAGdosage combinations. 30-100 100-150 150-200 200-250 mg/m² mg/m² mg/m²mg/m² 17-AAG 17-AAG 17-AAG 17-AAG  0-20 mg/m² 0337 0338 0339 0340paclitaxel  20-40 mg/m² 0341 0342 0343 0344 paclitaxel  40-60 mg/m² 03450346 0347 0348 paclitaxel  60-80 mg/m² 0349 0350 0351 0352 paclitaxel 80-100 mg/m² 0353 0354 0355 0356 paclitaxel 100-120 mg/m² 0357 03580359 0360 paclitaxel 120-140 mg/m² 0361 0362 0363 0364 paclitaxel140-160 mg/m² 0365 0366 0367 0368 paclitaxel 160-180 mg/m² 0369 03700371 0372 paclitaxel

TABLE 10 Paclitaxel/17-AAG dosage combinations continued. 250-300300-350 350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG  0-20 mg/m² 0373 0374 0375 0376 paclitaxel  20-40 mg/m² 0377 03780379 0380 paclitaxel  40-60 mg/m² 0381 0382 0383 0384 paclitaxel  60-80mg/m² 0385 0386 0387 0388 paclitaxel  80-100 mg/m² 0389 0390 0391 0392paclitaxel 100-120 mg/m² 0393 0394 0395 0396 paclitaxel 120-140 mg/m²0397 0398 0399 0400 paclitaxel 140-160 mg/m² 0401 0402 0403 0404paclitaxel 160-180 mg/m² 0405 0406 0407 0408 paclitaxel

Where the present method involves the administration of 17-AAG anddocetaxel, a dosage regimen involving one or two administrations of thecombination per week or longer (e.g., every 3 weeks) is typical. Tables11 and 12 below show a number of docetaxel/17-AAG dosage combinations(i.e., dosage combinations 0409 to 0488). TABLE 11 Docetaxel/17-AAGdosage combinations. 30-100 100-150 150-200 200-250 mg/m² mg/m² mg/m²mg/m² 17-AAG 17-AAG 17-AAG 17-AAG  0-10 mg/m² 0409 0410 0411 0412docetaxel 10-20 mg/m² 0413 0414 0415 0416 docetaxel 20-30 mg/m² 04170418 0419 0420 docetaxel 30-40 mg/m² 0421 0422 0423 0424 docetaxel 40-50mg/m² 0425 0426 0427 0428 docetaxel 50-60 mg/m² 0429 0430 0431 0432docetaxel 60-70 mg/m² 0433 0434 0435 0436 docetaxel 70-80 mg/m² 04370438 0439 0440 docetaxel 80-90 mg/m² 0441 0442 0443 0444 docetaxel90-100 mg/m² 0445 0446 0447 0448 docetaxel

TABLE 12 Docetaxel/17-AAG dosage combinations continued. 250-300 300-350350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG 17-AAG 0-10 mg/m² 0449 0450 0451 0452 docetaxel 10-20 mg/m² 0453 0454 04550456 docetaxel 20-30 mg/m² 0457 0458 0459 0460 docetaxel 30-40 mg/m²0461 0462 0463 0464 docetaxel 40-50 mg/m² 0465 0466 0467 0468 docetaxel50-60 mg/m² 0469 0470 0471 0472 docetaxel 60-70 mg/m² 0473 0474 04750476 docetaxel 70-80 mg/m² 0477 0478 0479 0480 docetaxel 80-90 mg/m²0481 0482 0483 0484 docetaxel 90-100 mg/m² 0485 0486 0487 0488 docetaxel

Where the present method involves the administration of 17-AAG andepothilone D, a dosage regimen involving one or two administrations ofthe combination per week or longer (e.g., every 3 weeks) is typical.Tables 13 and 14 below show a number of epothilone D/17-AAG dosagecombinations (i.e., dosage combinations 0489 to 0558). TABLE 13Epothilone D/17-AAG dosage combinations. 30-100 100-150 150-200 200-250mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG 17-AAG  0-10 mg/m² 04890490 0491 0492 epothilone D 10-20 mg/m² 0493 0494 0495 0496 epothilone D20-30 mg/m² 0497 0498 0499 0500 epothilone D 30-40 mg/m² 0501 0502 05030504 epothilone D 40-50 mg/m² 0505 0506 0507 0508 epothilone D 50-60mg/m² 0509 0500 0501 0502 epothilone D 60-70 mg/m² 0503 0504 0505 0506epothilone D 70-80 mg/m² 0507 0508 0509 0510 epothilone D 80-90 mg/m²0511 0512 0513 0514 epothilone D 90-100 mg/m² 0515 0516 0517 0518epothilone D

TABLE 14 Epothilone D/17-AAG dosage combinations continued. 250-300300-350 350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG  0-10 mg/m² 0519 0520 0521 0522 epothilone D 10-20 mg/m² 05230524 0525 0526 epothilone D 20-30 mg/m² 0527 0528 0529 0530 epothilone D30-40 mg/m² 0531 0532 0533 0534 epothilone D 40-50 mg/m² 0535 0536 05370538 epothilone D 50-60 mg/m² 0539 0540 0541 0542 epothilone D 60-70mg/m² 0543 0544 0545 0546 epothilone D 70-80 mg/m² 0547 0548 0549 0550epothilone D 80-90 mg/m² 0551 0552 0553 0554 epothilone D 90-100 mg/m²0555 0556 0557 0558 epothilone D

Where the present method involves the administration of 17-AAG andetoposide, a dosage regimen involving more than one or twoadministrations of the combination per week or is typical. Oftentimesthe combination is administered 3, 4 or 5 times per week. Tables 15 and16 below show a number of etoposide/17-AAG dosage combinations (i.e.,dosage combinations 0559 to 0654). TABLE 15 Etoposide/17-AAG dosagecombinations. 30-100 100-150 150-200 200-250 mg/m² mg/m² mg/m² mg/m²17-AAG 17-AAG 17-AAG 17-AAG  0-10 mg/m² 0559 0560 0561 0562 etoposide 10-20 mg/m² 0563 0564 0565 0566 etoposide  20-30 mg/m² 0567 0568 05690570 etoposide  30-40 mg/m² 0571 0572 0573 0574 etoposide  40-50 mg/m²0575 0576 0577 0578 etoposide  50-60 mg/m² 0579 0580 0581 0582 etoposide 60-70 mg/m² 0583 0584 0585 0586 etoposide  70-80 mg/m² 0587 0588 05890590 etoposide  80-90 mg/m² 0591 0592 0593 0594 etoposide  90-100 mg/m²0595 0596 0597 0598 etoposide 100-110 mg/m² 0599 0600 0601 0602etoposide 110-120 mg/m² 0603 0604 0605 0606 etoposide

TABLE 16 Etoposide/17-AAG dosage combinations continued. 250-300 300-350350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG 17-AAG 0-10 mg/m² 0607 0608 0609 0610 etoposide  10-20 mg/m² 0611 0612 06130614 etoposide  20-30 mg/m² 0615 0616 0617 0618 etoposide  30-40 mg/m²0619 0620 0621 0622 etoposide  40-50 mg/m² 0623 0624 0625 0626 etoposide 50-60 mg/m² 0627 0628 0629 0630 etoposide  60-70 mg/m² 0631 0632 06330634 etoposide  70-80 mg/m² 0635 0636 0637 0638 etoposide  80-90 mg/m²0639 0640 0641 0642 etoposide  90-100 mg/m² 0643 0644 0645 0646etoposide 100-110 mg/m² 0647 0648 0649 0650 etoposide 110-120 mg/m² 06510652 0653 0654 etoposide

Where the present method involves the administration of 17-AAG andteniposide, a dosage regimen involving more than one or twoadministrations of the combination per week or is typical. Oftentimesthe combination is administered 3, 4 or 5 times per week. Tables 17 and18 below show a number of teniposide/17-AAG dosage combinations (i.e.,dosage combinations 0655 to 0742). TABLE 17 Teniposide/17-AAG dosagecombinations. 30-100 100-150 150-200 200-250 mg/m² mg/m² mg/m² mg/m²17-AAG 17-AAG 17-AAG 17-AAG  0-15 mg/m² 0655 0656 0657 0658 teniposide 15-30 mg/m² 0659 0660 0661 0662 teniposide  30-45 mg/m² 0663 0664 06650666 teniposide  45-60 mg/m² 0667 0668 0669 0670 teniposide  60-75 mg/m²0671 0672 0673 0674 teniposide  75-90 mg/m² 0675 0676 0677 0678teniposide  90-105 mg/m² 0679 0680 0681 0682 teniposide 105-120 mg/m²0683 0684 0685 0686 teniposide 120-135 mg/m² 0687 0688 0689 0690teniposide 135-150 mg/m² 0691 0692 0693 0694 teniposide 150-165 mg/m²0695 0696 0697 0698 teniposide

TABLE 18 Teniposide/17-AAG dosage combinations continued. 250-300300-350 350-400 400-450 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG17-AAG  0-15 mg/m² 0699 0700 0701 0702 teniposide  15-30 mg/m² 0703 07040705 0706 teniposide  30-45 mg/m² 0707 0708 0709 0710 teniposide  45-60mg/m² 0711 0712 0713 0714 teniposide  60-75 mg/m² 0715 0716 0717 0718teniposide  75-90 mg/m² 0719 0720 0721 0722 teniposide  90-105 mg/m²0723 0724 0725 0726 teniposide 105-120 mg/m² 0727 0728 0729 0730teniposide 120-135 mg/m² 0731 0732 0733 0734 teniposide 135-150 mg/m²0735 0736 0737 0738 teniposide 150-165 mg/m² 0739 0740 0741 0742teniposide

The method of the present invention may be carried out in at least twobasic ways. A subject may first be treated with a dose on an HSP90inhibitor and subsequently be treated with a dose of an antimitotic.Alternatively, the subject may first be treated with a dose of anantimitotic and subsequently be treated with a dose of an HSP90inhibitor. The appropriate dosing regimen depends on the particularantimitotic employed.

In another aspect of the invention, a subject is first treated with adose of a an antimitotic (e.g., docetaxel, viblastine, vincristine,vindesine, or epothilone D). After waiting for a period of timesufficient to allow development of a substantially efficacious responseof the antimitotic, a formulation comprising a synergistic dose of abenzoquinone ansamycin together with a second sub-toxic dose of theantimitotic is administered. In general, the appropriate period of timesufficient to allow development of a substantially efficacious responseto the antimitotic will depend upon the pharmacokinetics of theantimitotic, and will have been determined during clinical trials oftherapy using the antimitotic alone. In one embodiment of the invention,the period of time sufficient to allow development of a substantiallyefficacious response to the antimitotic is between about 1 hour and 96hours. In another aspect of the invention, the period of time sufficientto allow development of a substantially efficacious response to theantimitotic is between about 2 hours and 48 hours. In another embodimentof the invention, the period of time sufficient to allow development ofa substantially efficacious response to the antimitotic is between about4 hours and 24 hours.

In another aspect of the invention, a subject is treated first with oneof the above-described benzoquinone ansamycins, and second, a dose of anantimitotic, such as, but not limited to, docetaxel, vinblastine,vincristine, vindesine and epothilone D. After waiting for a period oftime sufficient to allow development of a substantially efficaciousresponse of the antimitotic, a formulation comprising a synergistic doseof a benzoquinone ansamycin together with a second sub-toxic dose of theantimitotic is administered. In general, the appropriate period of timesufficient to allow development of a substantially efficacious responseto the antimitotic will depend upon the pharmacokinetics of theantimitotic, and will have been determined during clinical trials oftherapy using the antimitotic alone. In one embodiment of the invention,the period of time sufficient to allow development of a substantiallyefficacious response to the antimitotic is between about 1 hour and 96hours. In another aspect of the invention, the period of time sufficientto allow development of a substantially efficacious response to theantimitotic is between about 2 hours and 48 hours. In another embodimentof the invention, the period of time sufficient to allow development ofa substantially efficacious response to the antimitotic is between about4 hours and 24 hours.

As noted above, the combination of an HSP90 inhibitor and an antimitoticallows for the use of a lower therapeutic dose of the antimitotic forthe treatment of cancer. That a lower dose of antimitotic is usedoftentimes lessens the side effects observed in a subject. The lessenedside effects can be measured both in terms of incidence and severity.Severity measures are provided through a grading process delineated bythe National Cancer Institute (common toxicity criteria NCI CTC, Version2). For instance, the incidence of side effects are typically reduced10%. Oftentimes, the incidence is reduced 20%, 30%, 40% or 50%.Furthermore, the incidence of grade 3 or 4 toxicities for more commonside effects associated with antimitotic administration (e.g., anemia,anorexia, diarrhea, fatigue, nausea and vomiting) is oftentimes reduced10%, 20%, 30%, 40% or 50%.

Formulations used in the present invention may be in any suitable form,such as a solid, semisolid, or liquid form. See Pharmaceutical DosageForms and Drug Delivery Systems, 5^(th) edition, Lippicott Williams &Wilkins (1991), incorporated herein by reference. In general thepharmaceutical preparation will contain one or more of the compounds ofthe present invention as an active ingredient in admixture with anorganic or inorganic carrier or excipient suitable for external,enteral, or parenteral application. The active ingredient may becompounded, for example, with the usual non-toxic, pharmaceuticallyacceptable carriers for tablets, pellets, capsules, suppositories,pessaries, solutions, emulsions, suspensions, and any other formsuitable for use. The carriers that can be used include water, glucose,lactose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea, and other carriers suitable for use in manufacturingpreparations in solid, semi-solid, or liquefied form. In addition,auxiliary stabilizing, thickening, and coloring agents and perfumes maybe used. Where applicable, the compounds useful in the methods of theinvention may be formulated as microcapsules and nanoparticles. Generalprotocols are described, for example, by Microcapsules and Nanoparticlesin Medicine and Pharmacy by Max Donbrow, ed., CRC Press (1992) and byU.S. Pat. Nos. 5,510,118, 5,534,270 and 5,662,883 which are allincorporated herein by reference. By increasing the ratio of surfacearea to volume, these formulations allow for the oral delivery ofcompounds that would not otherwise be amenable to oral delivery. Thecompounds useful in the methods of the invention may also be formulatedusing other methods that have been previously used for low solubilitydrugs. For example, the compounds may form emulsions with vitamin E, ora PEGylated derivative thereof as described by PCT publications WO98/30205 and WO 00/71163, each of which is incorporated herein byreference. Typically, the compound useful in the methods of theinvention is dissolved in an aqueous solution containing ethanol(preferably less than 1% w/v). Vitamin E or a PEGylated-vitamin E isadded. The ethanol is then removed to form a pre-emulsion that can beformulated for intravenous or oral routes of administration. Anothermethod involves encapsulating the compounds useful in the methods of theinvention in liposomes. Methods for forming liposomes as drug deliveryvehicles are well known in the art. Suitable protocols include thosedescribed by U.S. Pat. Nos. 5,683,715, 5,415,869, and 5,424,073 whichare incorporated herein by reference relating to another relatively lowsolubility cancer drug paclitaxel and by PCT Publication WO 01/10412which is incorporated herein by reference relating to epothilone B. Ofthe various lipids that may be used, particularly preferred lipids formaking encapsulated liposomes include phosphatidylcholine andpolyethyleneglycol-derivatized distearyl phosphatidyl-ethanoloamine.

Yet another method involves formulating the compounds useful in themethods of the invention using polymers such as biopolymers orbiocompatible (synthetic or naturally occurring) polymers. Biocompatiblepolymers can be categorized as biodegradable and non-biodegradable.Biodegradable polymers degrade in vivo as a function of chemicalcomposition, method of manufacture, and implant structure. Illustrativeexamples of synthetic polymers include polyanhydrides, polyhydroxyacidssuch as polylactic acid, polyglycolic acids and copolymers thereof,polysters, polyamides, polyorthoesters and some polyphosphazenes.Illustrative examples of naturally occurring polymers include proteinsand polysaccharides such as collagen, hyaluronic acid, albumin, andgelatin.

Another method involves conjugating the compounds useful in the methodsof the invention to a polymer that enhances aqueous solubility. Examplesof suitable polymers include polyethylene glycol, poly-(d-glutamicacid), poly-(1-glutamic acid), poly-(1-glutamic acid), poly-(d-asparticacid), poly-(1-aspartic acid) and copolymers thereof. Polyglutamic acidshaving molecular weights between about 5,000 to about 100,000 arepreferred, with molecular weights between about 20,000 and 80,000 beingmore preferred wand with molecular weights between about 30,000 and60,000 being most preferred. The polymer is conjugated via an esterlinkage to one or more hydroxyls of an inventive geldanamycin using aprotocol as essentially described by U.S. Pat. No. 5,977,163 which isincorporated herein by reference.

In another method, the compounds useful in the methods of the inventionare conjugated to a monoclonal antibody. This method allows thetargeting of the inventive compounds to specific targets. Generalprotocols for the design and use of conjugated antibodies are describedin Monoclonal Antibody-Based Therapy of Cancer by Michael L. Grossbard,ED. (1998), which is incorporated herein by reference.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thesubject treated and the particular mode of administration. For example,a formulation for intravenous use comprises an amount of the inventivecompound ranging from about 1 mg/mL to about 25 mg/mL, preferably fromabout 5 mg/mL, and more preferably about 10 mg/mL. Intravenousformulations are typically diluted between about 2 fold and about 30fold with normal saline or 5% dextrose solution prior to use.

Preferably, 17-AAG is formulated as a pharmaceutical solutionformulation comprising 17-AAG in an concentration of up to 15 mg/mLdissolved in a vehicle comprising (i) a first component that is ethanol,in an amount of between about 40 and about 60 volume %; (ii) a secondcomponent that is a polyethoxylated castor oil, in an amount of betweenabout 15 to about 50 volume %; and (iii) a third component that isselected from the group consisting of propylene glycol, PEG 300, PEG400, glycerol, and combinations thereof, in an amount of between about 0and about 35 volume %. The aforesaid percentages are volume/volumepercentages based on the combined volumes of the first, second, andthird components. The lower limit of about 0 volume % for the thirdcomponent means that it is an optional component; that is, it may beabsent. The pharmaceutical solution formulation is then diluted intowater to prepare a diluted formulation containing up to 3 mg/mL 17-AAG,for intravenous formulation.

Preferably, the second component is Cremophor EL and the third componentis propylene glycol. In an especially preferred formulation, thepercentages of the first, second, and third components are 50%, 20-30%,and 20-30%, respectively.

Other formulations designed for 17-AAG are described in Tabibi et al.,U.S. Pat. No. 6,682,758 B1 (2004) and Ulm et al., WO 03/086381 A1(2003); the disclosures of which are incorporated herein by reference.

The method of the present invention is used for the treatment of cancer.In one embodiment, the methods of the present invention are used totreat cancers of the head and neck, which include, but are not limitedto, tumors of the nasal cavity, paranasal sinuses, nasopharynx, oralcavity, oropharynx, larynx, hypopharynx, salivary glands, andparagangliomas. In another embodiment, the compounds of the presentinvention are used to treat cancers of the liver and biliary tree,particularly hepatocellular carcinoma. In another embodiment, thecompounds of the present invention are used to treat intestinal cancers,particularly colorectal cancer. In another embodiment, the compounds ofthe present invention are used to treat ovarian cancer. In anotherembodiment, the compounds of the present invention are used to treatsmall cell and non-small cell lung cancer. In another embodiment, thecompounds of the present invention are used to treat breast cancer. Inanother embodiment, the compounds of the present invention are used totreat sarcomas, including fibrosarcoma, malignant fibrous histiocytoma,embryonal rhabdomyosarcoma, leiomyosarcoma, neuro-fibrosarcoma,osteosarcoma, synovial sarcoma, liposarcoma, and alveolar soft partsarcoma. In another embodiment, the compounds of the present inventionare used to treat neoplasms of the central nervous systems, particularlybrain cancer. In another embodiment, the compounds of the presentinvention are used to treat lymphomas which include Hodgkin's lymphoma,lymphoplasmacytoid lymphoma, follicular lymphoma, mucosa-associatedlymphoid tissue lymphoma, mantle cell lymphoma, B-lineage large celllymphoma, Burkitt's lymphoma, and T-cell anaplastic large cell lymphoma.

EXAMPLES

The following Examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in practicing theinvention.

Materials and Methods

Cell Line and Reagents

Human colon adenocarcinoma cell line, DLD-1, and human breastadenocarcinoma cell line, SKBr-3, were obtained from American TypeCulture Collection (manassas, Va.). DLD-1 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum, and SKBr-3 cellswere cultured in McCoy's 5a medium supplemented with 10% fetal bovineserum. 17-DMAG and 17-AAG were obtained using published procedures.Other cytotoxic agents were purchased commercially from suppliers suchas Sigma Chemical Co. (St. Louis, Mo.) and Sequoia Research Products(Oxford, UK).

Cell Viability Assay and Combination Effect Analysis

Cells were seeded in duplicate in 96-well microtiter plates at a densityof 5,000 cells per well and allowed to attach overnight. Cells weretreated with 17-AAG or 17-DMAG and the corresponding antimitotic atvarying concentrations, ranging from 0.5 picomolar (“pM”) to 50micromolar (“μM”), for 3 days. Cell viability was determined using theMTS assay (Promega). For the drug combination assay, cells were seededin duplicate in 96-well plates (5,000 cells/well). After an overnightincubation, cells were treated with drug alone or a combination and theIC₅₀ value (the concentration of drug required to inhibit cell growth by50%) was determined. Based on the IC₅₀ values of each individual drug,combined drug treatment was designed at constant ratios of two drugs,i.e., equivalent to the ratio of their IC₅₀. Two treatment scheduleswere used: In one schedule, the cells were exposed to 24 hours of 17-AAGor 17-DMAG. The drug was then added to the cells and incubated for 48hours. In another schedule, cells were exposed to the drug alone for 24hours followed by addition of 17-AAG or 17-DMAG for 48 hours. Cellviability was determined by the MTS assay.

Synergism, additivity or antagonism was determined by median effectanalysis using the combination index (CI) calculated using Calcusyn(Biosoft, Cambridge, UK). The combination index is defined as follows:CI=[D]₁/[D_(x)]₁+[D]₂/[D_(x)]₂

The quantities [D]₁ and [D]₂ represent the concentrations of the firstand second drug, respectively, that in combination provide a response ofx % in the assay. The quantities [D_(x)]₁ and [D_(x)]₂ represent theconcentrations of the first and second drug, respectively, that whenused alone provide a response of x % in the assay. Values of CI<1, CI=1,and CI>1 indicated drug-drug synergism, additivity, and antagonismrespectively (Chou and Talalay 1984). The “enhancing” effect of twodrugs can also be determined.

Results

17-AAG Combination in DLD-1 Cells

The following table provides CI values for combinations of 17-AAG andthe antimitotics docetaxel, vinblastine, vincristine, vindesine, andepothilone D in a DLD-1 cell assay. “Pre-administration” refers to theadministration of 17-AAG to the cells before the administration ofantimitotic; “post-administration” refers to the administration of17-AAG to the cells after the administration of antimitotic. TABLE 5 CIvalues for combinations in DLD-1 cells (human colorectal cancer cells).17-AAG 17-AAG Antimitotic Pre-Administration Post-AdministrationDocetaxel 0.79 ± 0.15 0.29 ± 0.12 Vinblastine 0.92 ± 0.3  0.38 ± 0.11Vincristine 0.96 ± 0.3  0.42 ± 0.08 Vindesine 0.91 ± 0.41 0.68 ± 0.08Epothilone D 0.84 ± 0.06 0.61 ± 0.12

17-AAG Combination in SKSBr-3 Cells

The following table provides CI values for combinations of 17-AAG andthe antimitotics docetaxel, vinblastine, vincristine, and epothilone Din an SKBr-3 cell assay. TABLE 6 CI values for combinations in SKBrcells (human breast cancer cells). 17-AAG 17-AAG AntimitoticPre-Administration Post-Administration Docetaxel 0.53 ± 0.11 0.59 ± 0.26Vinblastine 0.39 ± 0.03 0.68 ± 0.41 Vincristine 0.67 ± 0.37 0.99 ± 0.73Epothilone D 0.72 ± 0.07  0.58 ± 0.009

Additional Observations

Additional analysis indicated that both 17-AAG and 17-DMAG reduced theexpression of ErbB2 protein in SKBr3 and glioma cells. This observation,taken in combination with the results reported above, indicates thatcombinations of 17-AAG or 17-DMAG with any of the antimitotics abovethat are known to be useful to treat diseases characterized by elevatedErbB2 protein expression (i.e., levels of expressions of ErbB2 proteingreater than those found in healthy cells).

1. A method for treating breast cancer in a patient, wherein the methodcomprises administering an HSP90 inhibitor and an antimitotic to thepatient.
 2. The method of claim 1, wherein the HSP90 inhibitor isadministered to the patient before the antimitotic.
 3. The method ofclaim 1, wherein the HSP90 inhibitor is administered to the patientafter the antimitotic.
 4. The method of claim 2, wherein the HSP90inhibitor is geldanamycin or a geldanamycin derivative.
 5. The method ofclaim 3, wherein the HSP90 inhibitor is geldanamycin or a geldanamycinderivative.
 6. The method of claim 4, wherein the HSP90 inhibitor is ageldanamycin derivative, and wherein the derivative is 17-AAG.
 7. Themethod of claim 5, wherein the HSP 90 inhibitor is a geldanamycinderivative, and wherein the derivative is 17-AAG.
 8. The method of claim6, wherein the antimitotic is docetaxel, vinblastine, or epothilone D.9. The method of claim 7, wherein the antimitotic is epothilone D.
 10. Amethod for treating colorectal cancer in a patient, wherein the methodcomprises administering an HSP90 inhibitor and an antimitotic to thepatient.
 11. The method of claim 10, wherein the HSP90 inhibitor isadministered to the patient after the antimitotic.
 12. The method ofclaim 10, wherein the HSP90 inhibitor is administered to the patientbefore the antimitotic.
 13. The method of claim 11, wherein the HSP90inhibitor is geldanamycin or a geldanamycin derivative.
 14. The methodof claim 12, wherein the HSP90 inhibitor is geldanamycin or ageldanamycin derivative.
 15. The method of claim 13, wherein the HSP90inhibitor is a geldanamycin derivative, and wherein the derivative is17-AAG.
 16. The method of claim 14, wherein the HSP90 inhibitor is ageldanamycin derivative, and wherein the derivative is 17-AAG.
 17. Themethod of claim 1, wherein the HSP90 inhibitor is 17-AAG, and whereinthe administration of 17-AAG and the enzyme inhibitor is performed onceper week.
 18. The method of claim 1, wherein the HSP90 inhibitor is17-AAG, and wherein the administration of 17-AAG and the enzymeinhibitor is performed twice per week.
 19. The method of claim 10,wherein the HSP90 inhibitor is 17-AAG, and wherein the administration of17-AAG and the enzyme inhibitor is performed once per week.
 20. Themethod of claim 10, wherein the HSP90 inhibitor is 17-AAG, and whereinthe administration of 17-AAG and the enzyme inhibitor is performed twiceper week.
 21. The method of claim 17, wherein the therapeutic dose of17-AAG is between 50 mg/m² and 450 mg/m².
 22. The method of claim 18,wherein the therapeutic dose of 17-AAG is between 50 mg/m² and 250mg/m².
 23. The method of claim 19, wherein the therapeutic dose of17-AAG is between 50 mg/m² and 450 mg/m².
 24. The method of claim 20,wherein the therapeutic dose of 17-AAG is between 50 mg/m² and 250mg/m².